Characterization of TR‐107, a novel chemical activator of the human mitochondrial protease ClpP

Abstract We recently described the identification of a new class of small‐molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small‐molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR‐107) with excellent potency, specificity, and drug‐like properties for further evaluation. TR‐107 showed ClpP‐dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple‐negative breast cancer (TNBC) cell models. TR‐107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time‐, dose‐, and ClpP‐dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR‐107 exposure. The pharmacokinetic properties of TR‐107 were compared with other known ClpP activators including ONC201 and ONC212. TR‐107 displayed excellent exposure and serum t 1/2 after oral administration. Using human TNBC MDA‐MB‐231 xenografts, the antitumor response to TR‐107 was investigated. Oral administration of TR‐107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.


| INTRODUC TI ON
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is associated with poor prognosis, shorter progression-free, and overall survival compared with other breast cancer subtypes. [1][2][3] Unlike other subtypes, targeted therapies (e.g., tamoxifen and trastuzumab) are ineffective against TNBC leaving patients with limited options of systemic chemotherapy, surgery, or radiation, 1 or more recently immunotherapy. 4,5 As an alternative, considerable interest has developed in targeting mitochondrial metabolism as a potential approach to treat recalcitrant cancers. 6,7 Multiple studies now support the feasibility of targeting essential mitochondrial processes such as oxidative phosphorylation (OXPHOS), mitoribosomal activity, and TCA cycle function as a unique strategy to inhibit cancer cell proliferation. 8,9 This has led to the development of small-molecule inhibitors of OXPHOS, TCA cycle enzymes (isocitrate dehydrogenases [IDH1/2], α-ketoglutarate dehydrogenase), as well as modifiers of mitochondrial transcription (POLRMT) and lipid metabolism. 10,11 Included in this class of molecules are metformin and other clinical agents (BAY 87-2243, IACS-010759) designed to inhibit mitochondrial multimeric complex I enzyme that comprises the first step in the electron respiratory chain. [12][13][14] While these have shown promising activities in preclinical studies, the clinical application of these agents has yet to be realized. 9 The small-molecule imipridone ONC201 was initially identified in a screen for TRAIL inducers and later shown to have antagonistic effects on the dopamine receptors D2/D3. 15,16 While the direct target was unresolved, multiple studies showed that ONC201 exhibited anticancer responses in diverse cancer models including breast, pancreatic, leukemia, and others [16][17][18][19][20][21][22] leading to the clinical advancement of ONC201. The unexpected finding that ONC201 potently affected OXPHOS and mitochondrial metabolism in models of TNBC suggested an alternative mechanism of action. 23 Importantly, the development of the chemically related TR compounds (Madera Therapeutics), including immobilizable analogs, facilitated the discovery of the mitochondrial matrix protease ClpP as the major target. 24 Additional studies confirmed these findings and demonstrated that the activation of ClpP was essential for the anticancer properties of ONC201 and related analogs. 25,26 Modifiers of ClpP activity have previously shown anticancer properties, 27,28 with both inhibitors and activators of ClpP demonstrating antitumor efficacy for acute myelogenous leukemia. 29,30 Chemical activators of ClpP include diverse chemical structures such as the macrocyclic acyldepsipeptides (ADEPs) and D9 ( Figure S1A). ADEP and D9 were both described in 2018 as human ClpP activating agents prior to the discovery of a similar mechanism of action for ONC201 and the TR compounds. 27 Madera was the first to prepare a number of highly potent, cell-permeable ClpP activators through modifications of peripheral functionality on the chemical core of ONC201, and later through changes to the chemical core. 24 Detailed crystal structure analysis of ClpP and ONC201 further validated ClpP as a direct and specific binding partner. 25 ClpP is a component of the ClpXP protein complex localized in the mitochondrial matrix. ClpP is a tetradecameric serine protease that forms a complex with hexameric AAA+ ClpX, an ATPdependent protein unfoldase that enables substrate recognition and unfolding prior to its degradation by ClpP. [30][31][32][33] The crystal structure of ClpXP has been solved from a number of species providing detailed insight into how this proteolytic protein complex is regulated. ClpX binds to a hydrophobic pocket in ClpP thereby facilitating the opening of the axial pore and passage of unfolded proteins into the central barrel of ClpP. Pharmacological activators of ClpP, such as the ADEPs, were the first small molecules shown to bind to this hydrophobic pocket and open the axial pore of ClpP in the absence of ClpX, allowing for nonspecific entry of proteins into the active site of ClpP. 28,34,35 As part of the mitochondrial unfolded protein response, ClpXP canonically targets misfolded proteins to prevent the formation of experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212.
TR-107 displayed excellent exposure and serum t 1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

K E Y W O R D S
agonist, cell proliferation, mitochondria, oxidative phosphorylation, protease, small molecule, triple-negative breast cancer protein aggregates in the mitochondria. 31,36 ClpXP also has regulatory roles in heme biosynthesis, 37-39 mitophagy, 40,41 and reduction in reactive oxygen species levels following mitochondrial depolarization. 33 ClpP is overexpressed in breast cancer, 42 providing the potential for utilizing highly selective and potent ClpP activators as a novel approach to disrupt mitochondrial metabolic processes required for TNBC proliferation.
In this study, we present data on the characterization of a novel class of highly potent and selective ClpP activators. We focused on one compound, TR-107, and show that it induces the downregulation of selected mitochondrial proteins, impairs OXPHOS, and inhibits TNBC growth in a ClpP-dependent manner, with markedly 1 μg/ml hydrocortisone, and 1% anti/anti. Cells were maintained at 5% CO 2 and 37°C and periodically tested for mycoplasma.

| CRISPRi cell lines
sgRNA against human ClpP (Eurofins Genomics) was annealed and ligated into vector VDB783. This vector was transformed into DH5α cells and the presence of sgRNA was confirmed by colony PCR.
Lentivirus was produced in HEK293T cells following transfection with plasmid and jetPRIME (PolyPlus) as previously described. 43 Clonal populations of SUM159 cells infected with dCas9-KRAB lentivirus were clonally isolated to ensure dCas9-KRAB expression.

| ClpP activity assay
Measurement of in vitro activity of purified recombinant human ClpP was performed with a slight modification to the method previously described. 46 Briefly, ClpP activity was measured through the degra-

| Surface plasmon resonance
For the surface plasmon resonance (SPR) experiments, recombinant human ClpP was purified as previously described. 46 SPR measurements were performed on a Biacore X100 instrument (Cytiva Life Sciences) at 25°C in buffer (25 mM sodium phosphate, pH 7.5, 200 mM KCl, 0.05% Tween-20, and 0.004% DMSO). ClpP was immobilized onto flowcell two of a CM5 chip (Cytive Life Sciences) using the amine coupling wizard using Biacore X100 Control Software. A pulse of a mixture of NHS and EDC activated the CM5 chip surface.
ClpP was diluted to 100 μg/ml in 10 mM sodium acetate, pH 4.5 immediately prior to use and injected over the activated surface for 180 s. The remaining activated sites were blocked with a pulse of ethanolamine. 12 000 RU of ClpP was captured using this procedure. Data were blank subtracted and analysis was performed in Biacore X100 Evaluation Software (Cytiva Life Sciences). The steady-state response was calculated 15 s before the end of injection with a window of 15 s. Data were fitted to a one-site Langmuir binding model.

| Mitochondrial respiration analysis
Cellular oxygen consumption and extracellular acidification rates (OCR and ECAR, respectively) were measured using Seahorse XFe96

| Mitochondrial DNA (mtDNA) copy number analysis
Mitochondrial DNA copy number was determined as previously described. 23 Briefly, genomic DNA was isolated from tissue lysate with DNeasy Blood & Tissue kit (Qiagen, 69504). mtDNA copy number was determined by quantitative PCR with a human mitochondrial to nuclear DNA ratio kit (Takara Bio USA, 7246).

| Pharmacokinetic analysis
The compounds were evaluated for pharmacokinetic properties

| Protein-binding studies to plasma proteins
Protein-binding studies were performed by Eurofins Panlabs, Inc.
This assay utilizes equilibrium dialysis in a microplate format, as previously described. 47 Briefly, this analysis is used to determine the bound and unbound fraction of the drug and calculate the percentage of the test compound binding to murine plasma proteins.

| In vivo implantation and tumor measurement
MDA-MB-231 cells were harvested during exponential growth and resuspended in cold, sterile DPBS. Each animal received an orthotopic injection of 5 × 10 6 cells (0.05 ml volume) into the mammary fat pad.
Tumor growth was monitored as the average tumor size approached the target range of 60-100 mm 3 . Tumors were measured in two dimensions via caliper and volume was calculated using the following formula: where w is the width and l is the length (mm) of the tumor. Tumor weight was estimated based on the assumption that 1 mg = 1 mm 3 .
Fourteen days later (Day 1), animals were sorted into groups (n = 10 per group) with a mean tumor volume of 70-73 mm 3 .

| Treatment
Mice began dosing on Day 1 as summarized in Table S1. Compounds were administered orally (p.o.) in a dosing volume of 10 ml/kg. Twice daily doses (B.I.D) were administered at least 6 h apart.

| Endpoint and tumor growth delay analysis
Tumors were measured using calipers biweekly and animals were euthanized upon reaching tumor volume > 1500 mm 3 or on the final study day (Day 36), whichever came first. Time to endpoint (TTE) for analysis was calculated for animals that exited the study due to tumor volume using the following equation: where TTE is expressed in days, endpoint volume is expressed in Animals documented having died due to treatment-related deaths or nontreatment-related due to metastasis were assigned a TTE value equivalent to the day of death. Treatment outcome was evaluated from tumor growth delay (TGD) defined as the increase in the median TTE in the treatment group compared with the control group.

| Sampling
On Day 36, all animals in all groups were sampled. Tumors were excised, divided into two parts, and weighed. Part 1 was preserved in a 5 ml fixative solution (4% formaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer) and stored at 4°C. Part 2 was trimmed to be less than 0.5 cm in at least one dimension, submerged in five volumes of RNAlater solution, and stored at −20°C.

| Statistical analysis
Statistical analysis for viability, mitochondrial respiration, ClpP activity assays, and animal studies was performed using Prism 9 (GraphPad). Analysis of pharmacokinetic data was performed using FDA certified pharmacokinetic program Phoenix WinNonlin 7.0 (Pharsight). In the mouse xenograft study, study groups experiencing  Figure 1A, Figure S1A, or a previous publication. 24 We tested the TR compounds' ability to inhibit the growth of two

| Characterization of novel activators of ClpP
We examined the ability of the new TR compounds to bind and activate ClpP. Select TR compounds were incubated with purified recombinant human ClpP and ClpP proteolytic activity was deter-

| TR-107 inhibits TNBC growth without a significant increase in apoptosis
Since TR-107 showed excellent potency in these assays, this com-

| TR-107 induces time-and dose-dependent reduction of multiple mitochondrial proteins
We and others reported that treatment of MDA-MB-231 or ClpX, the ATPase subunit of the ClpXP complex, was previously observed to decline after 24 h of ONC212 treatment in pancreatic cancer models. 50 We observed ClpX decline rapidly (~3 h) after TR  Figure S2B). By comparison, the levels of these proteins were not affected in the ClpP-KO cell lines, even at the highest concentration of the compound tested ( Figure 3, Figure S2).
Heat shock protein 60 (HSP60) is a mitochondrial matrix chaperone required for maintaining the folding of imported proteins and HSP60 inhibitors are being investigated as potential anticancer compounds. 51 Immunoblotting for HSP60 protein after TR-107 treatment of MDA-MB-231 cells showed that HSP60 levels were significantly reduced in a time-dependent manner ( Figure S2D). DNA-directed RNA polymerase, mitochondrial (POLRMT) is a TFAM interacting protein involved in the replication of mitochondrial DNA. Recent studies have shown the efficacy of inhibiting POLRMT as a potential anticancer approach. 10 Because of the observed decline in TFAM, we investigated whether POLRMT was similarly affected by these treatments. Immunoblotting data showed that POLRMT followed a dose-dependent decline in fulllength protein level following TR compound treatment ( Figure S2E).
Together, these results demonstrate significant effects of TR-107 on mitochondrial proteins involved in energetic and homeostatic processes in a time-, dose-and ClpP-dependent manner.

| TR-107 inhibits OXPHOS in MDA-MB-231 cells
Because previous studies showed inhibition of OXPHOS by in extracellular acidification (ECAR, Figure 4C). Taken together, our results demonstrate substantial disruption of OXPHOS by TR107 in a dose-and ClpP-dependent manner.

As shown by Greer et al., ONC201 treatment resulted in a greater
dependence on glycolysis for cell survival, and switching media carbon sources from glucose to galactose enhanced cell growth inhibition by ONC201. 23 We similarly compared the effects of incubating

| Pharmacokinetic properties of TR-107
With kg or less in a murine model of breast cancer. Therefore, we chose to evaluate the oral exposure of these agents with a single dose of 10 mg/kg and in many cases a 2 mg/kg i.v dose allowing for an F% determination. Importantly, the results of our studies showed significant differences in PK properties among these compounds (Table 1).  (Table 1).
TR-57 and TR-107 displayed the highest exposure (AUC) of the tested agents when administered at 10 mg/kg orally (2710 and 2360 hr ng/ml respectively, Table 1). Both compounds showed rapid absorption via oral administration with an F% of 61% (TR-57).
In addition, protein-binding studies for TR-107 show a serum-free fraction of 10% (mouse, Figure S3C). Because of the favorable PK characteristics observed with TR-107 and its efficient molecular design, this compound was selected for advancement in animal xenograft studies described below.

| TR-107 inhibits tumor growth in an MD-MBA-231 mouse xenograft model
To determine the in vivo antitumor efficacy of TR-107, we examined dose-dependent responses using an MDA-MB-231 mouse xenograft model. MDA-MB-231 cells in Matrigel were orthotopically injected into the flank mammary fat pad of female NCr nu/nu mice (10/group) as described in Section 2 and Appendix S1. Vehicle control (Group 1) and TR-107 treatments (4 mg/kg [Group 2], 8 mg/kg [Group 3]) were administered by oral gavage at frequencies described in Table S1.
The results of these studies demonstrated a dose-and timedependent reduction in tumor volume. The mean tumor volume decreased for both treatment groups (G2, G3) with a ~50% reduction in tumor volume observed on day 26 ( Figure 5A). Comparing the two different groups over the course of the study did not reveal significant advantages of one dosing regimen over the other in tumor volume reduction ( Figure 5A,B) or survival ( Figure 5C). A ~35% increase in median survival was also observed in mice treated with TR-107 for both groups ( Figure 5C). Consistent with ClpP activation, analysis of the tumor lysates from control and treated animals (G3), showed a substantial loss of mitochondrial DNA ( Figure 5D). TR-107 treatment was well tolerated and less than 5% weight loss was observed even at the highest dosing regimen ( Figure S3). Effects of ONC201 and TR com-

| DISCUSS ION
ONC201, a screening hit with previously described CNS activity, 54 was the first imipridone molecule shown to be effective against a  [24][25][26] has redefined our understanding of the anticancer mechanism of these agents. Moreover, the identification of ClpP as the major target has enabled the design and characterization of highly potent and specific ClpP activators. In this study, we describe the advancement of the chemically diverse TR compounds as selective ClpP activators, which includes molecules of different chemical scaffolds from which agents for preclinical assessment were selected. We characterize one of these compounds, TR-107, for effects on mitochondrial metabolism and protein turnover, PK properties, and TNBC growth inhibition both in vitro and in vivo. Importantly, these studies further demonstrate the pivotal role of ClpP activation in the mechanism of TR-107 action.
From the collection of TR compounds, we were able to perform initial structure-activity relationship (SAR) studies using cell growth inhibition and ClpP activation as determinants. Nanjing Gator Meditech was the first to accurately determine that for the imipri- target identification studies. 24 We now report that further modifica- Because of its high potency in cell growth and ClpP activation assays, TR-107 was selected for further mechanistic, pharmacokinetic, and animal model studies. Other attributes include a low molecular weight (<400 amu), highly potent, and defined ClpP dependence, excellent PK properties, and efficacy against TNBC models both in vivo and in vitro. Using two independent ClpP-KO cell lines, our studies confirm the mitochondrial protease ClpP as the key target for TR-107 and related compounds. This includes effects on cell growth, OCR, and mitochondrial protein level as determined by immunoblotting. While ONC201, at concentrations far higher than that is necessary to activate ClpP or inhibit cell growth, has shown effects on dopamine receptors D2/D3, 55,59 our data 24 and that of others 23,25 strongly argues against a major role of the dopamine receptors in growth inhibition.
In strong support of a mitochondrial mechanism of action, we

| Limitations
There are a number of limitations in our studies that should be mentioned. The characterization of the direct effects of these compounds on purified recombinant ClpP may not accurately replicate the ClpP activation kinetics in vivo. This may be a factor of the concentration of ClpP, the artificial substrates or the assay conditions used that underestimate the potency of ClpP activation by these compounds. In fact, our data from the cell-based analysis of dose-dependent ClpP activation, very closely parallels the effects observed on growth inhibition, for both ONC201 and the TR compounds. Therefore, we believe that the cell-based assays are a better indicator of ClpP activation efficacy and further reflect potential differences in compound cell permeability as well.
While there are potential limitations with CRISPR-CAS generation of knockouts, the fact that we obtained similar results with two completely different CLPP knockout lines, strongly supports the specificity of the compound effects on ClpP. As with any animal study, there are also limitations to our animal studies. We performed our analysis with the least number of animals to achieve a statistically powered result. Given the fact that ONC201 has been extensively examined in other animal models, we only tested the effects of the TR compounds in these studies. While female mice were used exclusively in our experiments, we acknowledge that a similar study in male mice could be valuable. However, since the focus of our study was breast cancer, we believe that our rationale for this initial study was logical. Last, we acknowledge the limitations of studying a single xenograft model. Ideally, additional studies will be performed in syngeneic models of breast cancer to fully characterize these important new compounds. Der, S. Lipkowitz, and W. Houry.

ACK N OWLED G M ENT
The authors would like to acknowledge Dr. Michael P. East for invaluable scientific discussion and generous help with experimental troubleshooting.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data can be made available upon request from the authors.

D I SCLOS U R E S
CJD is a consultant/advisory board member for Anchiano Therapeutics,

Deciphera Pharmaceuticals, Mirati Therapeutics, and SpringWorks
Therapeutics and has consulted for Eli Lilly, Jazz Therapeutics, Ribometrix, Sanofi, and Turning Point Therapeutics. EJI and HL both have a financial interest in Madera Therapeutics.

E TH I C S S TATEM ENT
All animal experiments were performed in accordance with ethi-